How to measure and predict the molar absorption coefficient of a protein

Pace, C. Nick; Vajdos, F.F.; Fee, Lanette; Grimsley, Gerald R.; Gray, Tamara E.

doi:10.1002/pro.5560041120

Cited by 3,811 publications

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“…Frozen aliquots of scMN+8 were reconditioned to the correct buffer with a NAP-10 column (GE Healthcare, Buckinghamshire, U.K.). The protein concentrations were spectrophotometrically determined at 280 nm with the extinction coefficients 1490 M −1 cm −1 for CB, 67 14600 M −1 cm −1 for scMN, 68 and 3200 M −1 cm −1 for calmodulin. 69 To retain CB in the apo form, 200 μM EDTA was added to all buffers.…”

Section: ■ Methodsmentioning

confidence: 99%

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

Assarsson

Hellstrand

Cabaleiro-Lago

et al. 2014

ACS Chem. Neurosci.

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The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer's disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D 9k and singlechain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, −2, and −4, whereas no effect is observed for proteins with net charge of −6 and −8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D 9k and single-chain monellin bind to Aβ40.

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Section: ■ Methodsmentioning

confidence: 99%

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

Assarsson

Hellstrand

Cabaleiro-Lago

et al. 2014

ACS Chem. Neurosci.

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“…The protein concentration was determined using the extinction coefficient of ε = 9 800 M -1 cm -1 at 278 nm for RNase A and 9 940 M -1 cm -1 for the disulfide variants according to the calculations by Pace et al [43].…”

Section: Determination Of the Protein Concentrationmentioning

confidence: 99%

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Pecher¹,

Arnold²

2009

Biophysical Chemistry

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International audienceThe significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond

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“…The concentration of a pure protein may be determined by quantifying absorbance at 280 nm [16][17][18]. Most of the absorbance of proteins at this wavelength is due to the sidechains of the aromatic amino acids tryptophan and tyrosine.…”

Section: Exercise 4: Luxg-his 6 Concentration By Direct Spectrophotommentioning

confidence: 99%

Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi

Crowley

2010

Biochem Molecular Bio Educ

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In Photobacterium, the flavin reductase encoded by luxG regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a lightemitting reaction. A set of experiments, that employs a luxG-expression plasmid construct (pGhis) and is suitable for an undergraduate laboratory course, is presented. Hexahistidine-tagged protein is expressed in E. coli from pGhis, with the T7 RNA polymerase/lac repressor induction system. Bacteria are lysed by sonication and the tag allows for purification by immobilized metal ion affinity chromatography. A gel filtration column is used to remove ions and the other small molecules. The Bradford assay, with multiwell plates and an automated plate reader, is used to identify protein concentration peaks from both columns. The concentration of purified enzyme is then calculated from its A 280 using the predicted extinction coefficient. Yield and purity are further assayed with SDS-PAGE. Activity of purified enzyme is measured with riboflavin or FMN as substrate. Reaction rate is quantified by monitoring decrease in A 340 as the redox partner, NADH, is oxidized.Keywords: recombinant protein expression, immobilized metal ion affinity chromatography, LuxG, flavin reductase.Bioluminescence occurs in a variety of organisms; e.g., firefly, marine invertebrates and bacteria. Most of the luminescent bacteria exist in symbiosis with fish, proliferating within certain tissues and making the fish appear to be luminescent. The reaction that generates the light energy is catalyzed by the luciferase enzyme in all these organisms. However, the substrates of the reaction vary quite a bit. In the luminescent marine bacteria Photobacterium leiognathi and Photobacterium fischeri (previously known as Vibrio fischeri), one of the substrates is the fully-reduced, quinone form of flavin mononucleotide (FMN):As the light-generating reaction involves oxidation of FMNH 2 to FMN, sustained luminescence necessitates the recycling of FMN back to the reduced form. Such a reaction (Fig. 1) is usually catalyzed by a flavin reductase enzyme [2] (the reduced form is often shown as FMNH 2 , however at pH > 6.3 it ionizes to FMNH 2 [3]). NADH is oxidized as FMN is reduced.In P. fischeri and P. leiognathi the luciferase enzyme is an ab heterodimer [4]. The genes for these subunits, luxA and luxB, are encoded within the lux operon [5][6][7][8]. This operon also includes luxG that codes for a flavin reductase (protein is designated LuxG [9]). Thus, the translation of the lux operon-encoded mRNA should generate equimolar amounts of luciferase enzyme and flavin reductase.

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How to measure and predict the molar absorption coefficient of a protein

Cited by 3,811 publications

References 61 publications

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi

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